With the rapid recent growth of nanotechnology, convenient methods for the preparation, purification, isolation, modification, and other manipulations of nanoparticles are needed, particularly methods that are suitable for use to produce commercial quantities of nanoparticles of high purity and quality. For example, semiconductor nanocrystals are prepared in growth media containing a wide array of chemicals, including high-boiling solvents, alkylamines, alkyl phosphines, organic acids, phosphine oxides, phosphonic acids, and various salts, from which the nanocrystals must be isolated. After they are made, these nanocrystals are often treated with still more chemicals to modify their surface characteristics. For example, a semiconductor nanocrystal often is capped with a ‘shell’ that improves its optical characteristics, enhances its chemical stability, and optimizes other physical properties such as hydrophobicity, polarity, and solubility. In some cases, the shell makes the surface amenable to specific modifications that facilitate conjugation of the nanocrystal to another chemical species such as an antibody that can selectively deliver the conjugated nanocrystal to a particular type of receptor or cell.
That is, for many applications, nanoparticles must have certain types of functional groups on their surfaces to cause them to interact with or to become attached to a target molecule, particle, cell, or tissue. Methods for attaching suitable functional groups on the surfaces of nanoparticles are known in the art. However, for optimal performance and for many uses that demand highly consistent nanoparticles, there remains a need for methods to enhance the consistency of nanocrystal surface chemistry. For example, even if the nanocrystals in a batch are consistent in size, i.e., their size distribution is narrow enough to keep their optical properties consistent across a batch of nanocrystals, variations in surface chemistry may affect how well the nanocrystals perform in situations where impurities on the surface could be directly detrimental (e.g., for in vivo use, they could introduce toxicity concerns or promote problematic immunogenic responses) or indirectly detrimental (such as by interfering with efficient interactions with a target, or causing interactions to occur with non-target molecules, cells or tissues.) Particularly in applications where a single nanocrystal will be tracked, or where size differences that may be caused by differences in surface chemistry are important (e.g., where a nanoparticle is being observed via a FRET interaction that may make distance between the nanoparticle core and another absorber particularly important), having a highly uniform and very pure surface coating can be particularly important.
Such nanoparticles are made or modified using methods requiring complex reaction media. Frequently, nanocrystals such as quantum dots are made and/or modified while they are dissolved or suspended in a medium that contains high-boiling solvents, alkyl amines, alkyl phosphines, carboxylic acids, phosphonic acids, and other chemicals. This wide array of chemicals makes purification of the nanoparticle products challenging, particularly when the nanoparticles are to be used in applications where trace impurities carried with or adsorbed on the nanoparticles may interfere. Methods for the isolation and of nanoparticles from such complex media are needed that are quick and efficient, and that deliver a high yield of desired nanocrystal product with few or no deleterious impurities or contaminants.
Fluorescence-based analyses and nonisotopic detection systems have become a powerful tool for scientific research and clinical diagnostics for the detection of biomolecules using various assays including, but not limited to, flow cytometry, nucleic acid hybridization, DNA sequencing, nucleic acid amplification, immunoassays, histochemistry, and functional assays involving living cells. Fluorescent semiconductor nanocrystals have found widespread use due to their high fluorescent intensity and the ability of different nanocrystals to be excited by a single light source.
Peng et al. U.S. Pat. No. 6,872,249 disclose the synthesis of colloidal nanocrystals. A method of synthesizing colloidal nanocrystals is disclosed using metal oxides or metal salts as a precursor. The metal oxides or metal salts are combined with a ligand and then heated in combination with a coordinating solvent.
Peng et al. U.S. Pat. No. 6,869,545 discloses colloidal nanocrystals with high photoluminescence quantum yields and methods of preparing the same. The disclosure provides compositions containing colloidal nanocrystals with high photoluminescence quantum yields, synthetic methods for the preparation of highly luminescent colloidal nanocrystals, as well as methods to control the photoluminescent properties of these colloidal nanocrystals.
Bawendi et al. in U.S. Pat. No. 6,306,610 disclose quantum dot white and colored light emitting diodes (LEDs). The disclosure describes an electronic device comprising a population of quantum dots embedded in a host matrix and a primary light source which causes the dots to emit secondary light of a selected color, and a method of making such a device. The size distribution of the quantum dots is chosen to allow light of a particular color to be emitted from the structure. The dots can be composed of an undoped semiconductor such as CdSe, and may optionally be overcoated to increase photoluminescence. The host matrix for the device includes isolated dots within the matrix and not defined aggregates of nanocrystals.
U.S. Pub. No. 20040110220 to Mirkin et al. discloses nanoparticles having oligonucleotides attached to them and uses for such coated nanoparticles. The disclosure provides methods of detecting a nucleic acid that comprise contacting the nucleic acid with one or more types of nanoparticles having oligonucleotides attached to them. The disclosure describes a method where oligonucleotides are attached to nanoparticles and have sequences complementary to portions of the sequence of the nucleic acid. A detectable change is brought about as a result of the hybridization of the oligonucleotides on the nanoparticles to the nucleic acid. The disclosure describes methods of synthesizing nanoparticle-oligonucleotide conjugates and methods of using the conjugates. The disclosure describes nanomaterials and nanostructures comprising nanoparticles and methods of nanofabrication utilizing nanoparticles. The disclosure describes a method of separating a selected nucleic acid from other nucleic acids.
Nanocrystals are typically separated from their growth/preparation medium by the addition of polar solvents such as methanol. This method is often used where the nanocrystals are coated with a hydrophobic coating, because increasing the polarity of the growth medium by adding an alcohol causes the hydrophobic nanocrystals to precipitate from solution. Unfortunately, other hydrophobic materials in the growth medium can also precipitate, and in some cases, can be isolated in higher abundance than the desired nanocrystals. Extraction methods can be employed to separate the nanocrystals from the undesired hydrophobic material, but this usually reduces the yield and increases the cost of the product.
Multiple literature references describe the use of methanol to separate nanocrystals from growth media in this manner. These include Murray, C. B. et al., J. Am. Chem. Soc. 115: 8706 (1993); Hines, M. A. and Guyot-Sionnest, P., J. Phys. Chem. 100: 468 (1996); Peng, X. et al., J. Am. Chem. Soc. 119: 7019 (1997); and Dabbousi, B. O. et al., J. Phys. Chem. B. 101:9463 (1997).
In addition to the challenges of obtaining nanoparticles in high yields, nanoparticles can contain undesired excess oxidizable compounds on their surfaces. When nanoparticles are removed from their growth media by standard literature methods (precipitation with methanol), and then left to stand in solution, one frequently observes white solids that precipitate over time from the solution. Fluorescence measurements can be used to demonstrate that the precipitated solids are not nanocrystals. This demonstrates that the precipitation process using methanol may fail to remove certain impurities that are present in the complex media in which nanoparticles are typically formed and/or modified.
Accordingly, there is a need for improved methods suitable for isolation of nanoparticles from complex media, which ideally deliver high yields of desired product free of undesired impurities or contaminants, and which involve manipulations that are readily applied to large-scale nanoparticle preparation. Moreover, there is also a need for methods suitable for isolating or purifying nanocrystals made by known methods, which would ideally deliver high yields of clean and consistent nanoparticles, free of undesired impurities or contaminants, and which would involve manipulations that are readily applied to large-scale nanoparticle preparation.